The overall goal of this research is to improve methadone maintenance treatment, the cornerstone of opiate abuse therapy and a vitally effective strategy for HIV/AIDS risk reduction. Methadone disposition is characterized by extreme, unexplained and unpredictable inter- and intra- individual variability, causing opiate withdrawal, side effects, and treatment failures. Methadone intestinal first-pass metabolism and systemic clearance are catalyzed predominantly by cytochrome P4503A4. CYP3A4 variability and most importantly, CYP3A4 drug interactions, profoundly affect methadone first-pass metabolism and systemic clearance. Methadone is a newly recognized substrate for intestinal and CNS P-glycoprotein (P-gp), which determines methadone absorption and CNS pharmacodynamics in animals (a human role is unknown). The HIV/AIDS protease inhibitors (HIV-PI) and non-nucleoside reverse transcriptase inhibitors (NNRTI) are exquisitely potent CYP3A4 modulators, causing significant and complex (short- vs long-duration effects) yet poorly understood drug interactions. HIV-PI are P-gp modulators in vitro, yet their clinical effects are unknown. Unfortunate anecdote, the current way of detecting methadone drug interactions, has recently identified clinically significant HIV-PI and NNRTI-methadone interactions, with adverse outcomes. Nevertheless, such interactions are poorly understood. The overall research objective is to identify the mechanism(s) of HIV-PI and NNRTI-methadone interactions, and more generally validate a novel in vivo CYP3A4 probe for drug interactions involving HIV-PI, NNRTI, and methadone, and generalizable to other HIV/AIDS drugs, drug abuse therapies, and CYP3A4 drugs. The specific aims are to: 1) validate the pharmacodynamics of alfentanil (a highly specific in vivo CYP3A4 probe) as a rapid, noninvasive, inexpensive pharmacokinetic surrogate and probe for hepatic and intestinal CYP3A activity and drug interactions;2) determine the role of P-gp in methadone intestinal absorption and CNS pharmacodynamics in humans;3) determine HIV-PI (ritonavir, indinavir, saquinavir, nelfinavir)and NNRTI (nevirapine, efavirenz) effects on intestinal P-gp activity, first-pass CYP3A metabolism, and hepatic CYP3A activity;4) identify mechanisms of HIV-PI and NNRTI alterations in methadone disposition and clinical effect, potentially caused by modulation of P-gp-mediated intestinal absorption, CYP3A4-catalyzed first-pass metabolism, CYP3A-dependent systemic clearance, and/or P-gp- mediated CNS accessibility;5) establish the ability of noninvasive in vivo probe of CYP3A activity to predict methadone disposition, and to rapidly and noninvasively detect and predict drug interactions with HIV-PI, NNRTI, and methadone. Successful completion will provide fundamental new information on methadone disposition, improve the treatments and outcomes of opiate addiction and HIV/AIDS, and provide a novel technology for assessing CYP3A, the most important drug metabolism enzyme in humans.